Cell migration is a fundamental biological process that is required for numerous developmental, homeostatic and disease-related events. Within the immune system, cell migration is required to mediate appropriate immune cell development, generation of immune responses and effective protection against pathogens. Although we have a basic understanding of the migratory pathways followed by cellular populations, we know little about the migratory behavior of individual cells. In this application, we will focus on T cell migration out of tumors and the islets of diabetes-prone mice. We know that multiple cell types, including T cells, migrate into solid tumors and pancreatic islets, but we don't know if individual T cells migrate in and stay, transit in and out, or permanently remain outside once they have left. These represent just some of the numerous questions concerning cellular migration that pertain to developmental, homeostatic and disease-related events which remain obscure. This fundamental gap in our understanding is in part due to the complexity of tracking the migratory pathway of individual cells and determining their fate following their exit from restricted anatomical locations. The goal of this innovative R21 application is to develop genetic tools that will address this unmet need. Aim 1: BarTag: a Genetically-Encoded, In Vivo Single-Cell Migration Tracking System. We will develop a genetically-encoded mouse model in which T cells that migrate into defined anatomical locations will be marked with fluorescent tags that will facilitate analysis of their subsequent migratory pathway. We will use the high affinity barnase-barstar (KD - 10-14 M) as the receptor:ligand pair that will system. Thus, T cells expressing membrane-tagged barnase (the 'receptor') would enter a specific defined anatomical location that expresses tdTomato-barstar (the 'marker-tag'), which would bind to the infiltrating T cells thereby facilitating analysis of their subsequent migration. We will use E.G7, an OVA-expressing transfectant of the thymoma EL4, and OVA-specific CD8+ T cells from OTI TCR transgenic mice as an in vivo test system to determine the best 'marker-tag:receptor'combination. Aim 2: Defining T Cell Migration Dynamics from Restricted Anatomical Locations. We will develop and validate in vivo models for tracking cellular migration from defined anatomical locations, using B16 melanoma in C57BL/6 mice and pancreatic beta cells islets in diabetes-prone NOD mice as model systems. We will generate knockin/transgenic mouse models that will facilitate the expression of the 'receptor'and the 'marker- tag'on defined cell types and anatomical locations using Cre-lox recombination in C57BL/6 and NOD mice. These tools could then be used to answer numerous questions pertaining to the migration of multiple cell types in many normal and disease models. PUBLIC HEALTH RELEVANCE: Cell migration is a fundamental biological process that is required for numerous developmental, homeostatic and disease-related events. Although we have a basic understanding of the migratory pathways followed by cellular populations, we know little about the migratory behavior of individual cells. New tools are required to gain a greater understanding of the dynamics of cell migration as this may lead to new therapeutic interventions in cancer and autoimmune diseases, like type 1 diabetes, and enhanced vaccination protocols against a variety of pathogens.